Development of Innovational Brem Bali Products with Probiotics through Immobilization of Durian Skin Flour as a Healthy Alcoholic Beverage

The alcohol produced from the fermentation process in the traditional Balinese brem drink is one of the obstacles. Innovation and functional studies of brem Bali products are needed to be standardized and industrialized. The Bali brem fermentation process uses a dry starter, yeast, and a dominant growing microbe, Saccharomyces cerevisiae. The alcohol content in Bali brem ranges from 3-10%. Several studies have studied the probiotics in alcoholic beverages to provide health benefits potentially. In addition, durian skin flour is claimed to be used as a probiotic immobilizer. The development of brem drink with the addition of probiotics can have a major impact on health; in addition to the benefits of brem as a fermented drink, it also provides probiotic effects for health, including helping digestion as irritable bowel syndrome, reducing digestive tract symptoms (constipation, non-pathogenic diarrhoea, and flatulence), intolerance lactose, as the body’s defence (allergies such as eczema, atopic), cariogenic, carcinogenic, mutagenic, tumours, intestinal inflammation, intestinal bacterial overgrowth, lowering blood cholesterol, and hypertension.


Introduction
Sustainable Development Goals (SDGs) 2 represents a worldwide commitment to addressing the issues of undernutrition and hunger.The importance of enhancing nutritional quality is acknowledged within SDG 2, whose objective is to eradicate hunger, attain food security, promote improved nutrition, and support sustainable agriculture.This objective recognizes the notable strides made in combatting hunger and malnutrition since the year 2000.Nevertheless, the complete 1324 (2024) 012110 IOP Publishing doi:10.1088/1755-1315/1324/1/012110 2 elimination of hunger, food insecurity, and malnutrition for all necessitates sustained and targeted efforts, particularly in the regions of Asia and Africa.Moreover, the pursuit of improved nutrition extends beyond the confines of SDG 2. The achievement of nutritional goals is interconnected with all the Sustainable Development Goals (SDGs), as enhancing nutrition is the cornerstone of global sustainable development.Addressing malnutrition will yield far-reaching benefits in terms of health enhancement and poverty reduction.Consequently, making advancements in nutrition is indispensable for advancing sustainable development.
Brem is a fermented traditional food product.In Indonesia, there are two brem types: solid brem and liquid brem [1].Both types of brem have similarities in their production, namely using glutinous rice as the main ingredient.Solid brem is a traditional snack, fermented sticky rice with hardened yeast.In contrast to solid brem, liquid brem originating from Bali plays a role in religious ceremonies as a means.Based on the teachings of Hinduism in Bali and literature, it is explained that brem is a means of worshipping the Almighty God and His manifestations.
Bali brem drink was originally known as part of traditional and religious rituals on the Island of the Gods.Brem is used in traditional Hindu religious ceremonies in Bali as a substitute for blood in the Tabuh Rah ceremony or the Bhuta Yadnya ceremony.However, now its function is expanding, Brem Bali is starting to become known to the world as a "welcome drink" and is used as a unique souvenir from the island of Bali.In Indonesia, brem is better known as a traditional solid snack originating from Madiun and Wonogiri.At the same time, liquid brem is a typical drink of the Island of the Gods produced in Lombok.Liquid Brem, or rice wine, is made from black glutinous rice and white sticky rice through the stages of fermentation using a dry starter called yeast tape [2].Brem Bali is in great demand by tourists in Bali, most of whom come from Japan, the Netherlands, China and other European countries.Even now, Brem Bali has been widely exported to these countries.The existence of market opportunities not only in the country has resulted in the proliferation of home industries, such as MSMEs which have made Brem a business opportunity.Based on survey data, brem factories in Bali can produce 50-100 kg of waste daily or around 1.5-3 tons per month [3].
Liquid brem has a slightly sour sweet taste with an alcohol content of 3-10%; the alcohol content in brem can change during storage [3].The increase in alcohol content occurs due to the fermentation process, which continues during storage [4].Traditional drinks containing alcohol, such as brem, must be properly monitored and standardized.The risk of health effects that can be caused due to the alcohol content that is owned is not good if consumed in excess [4].This review intends to investigate innovation and functional assessment of brem Bali products to be used as a healthier choice of alcoholic beverages so Brem Bali can be standardized and industrialized globally.

Methods
References in the review study are articles from reputable national journals accredited from Sinta, reputable international journals accredited by Scopus from online web server publishers such as Taylor & Francis Online, Springer Link, Google Scholars, Wiley Online Library, and Science Direct.The tools used were Mendeley software [version 1.19.8 (2020)].Research literature was selected based on the screening of titles, and abstracts were analyzed to determine suitability with the selected inclusion and exclusion criteria.Inclusion criteria are a way of selecting reputable and internationally accredited articles.The selected articles are studies with data published within the last ten years (2013-2022).

Microbes that play an important role in the fermentation of Bali Brem
Liquid brem can be classified into two types: red brem, which is made from a mixture of white and black glutinous rice, and white brem, which is made from white glutinous rice [5].According to SNI 01-3952-1995, liquid brem is an alcoholic drink due to the fermentation process of glutinous rice (Oryza sativa glutinosa).Yeast is a microorganism that has an important role in the process of food fermentation; in the manufacture of Bali brem, some microbes dominantly grow and help the fermentation process, namely Saccharomyces cerevisiae, Candida glabrata, Pichia anomaly, and Orientalis issatchenkia [6].For thousands of years, the yeast Saccharomyces cerevisiae has become a microorganism widely used in making food and beverage products, especially those related to fermentation.
Fermentation as a biochemical modification mechanism is widely used in several food and beverage production processes, such as the conversion of sugars into alcoholic products, organic acids, carbon dioxide and flavoring compounds, as well as mediation processes by yeast and their enzymes.Fermentation can control microbial growth and improve taste, aroma, shelf life, texture, nutritional value of food, and product attractiveness [7].Saccharomyces cerevisiae has been widely used in fermentation processes and is closely related to fermented food and beverage products for consumption, such as bread, chocolate, wine and beer.In addition, this yeast also incorporates many desirable industrial properties, including a relatively high fermentation capacity (binding various sugars such as glucose, fructose, mannose, galactose, sucrose, and maltose), flocculation, ethanol tolerance, osmotic pressure, low pH tolerance, and it does not cause a high increase in oxygen in the fermentation process.Fermentative yeasts, particularly those classified as Saccharomyces cerevisiae, are resilient microorganisms utilized in food and beverage fermentation in diverse formats and under varying environmental conditions [7].The fermentation process can detoxify and destroy unwanted constituents in raw foods, such as phytates, tannins, polyphenols, and lactose removers.For example, cocoa beans cannot be eaten because they taste bitter and astringent.Still, after being fermented, they become the raw material for making chocolate [7].
Yeast production in Europe reaches 1 million tons annually, and about 30% of it is exported globally, with an annual global market growth rate of 8.8% from 2013 to 2018 [8].Saccharomyces cerevisiae is a unicellular fungus with a core genomic DNA of 12068 kilobases (kb) arranged in 16 chromosomes with the genome fully sequenced in 1996 by Goffeau and found to contain around 6000 genes, of which 5570 are thought to be protein-coding genes.Saccharomyces cerevisiae is a singlecelled, eukaryotic yeast-type microbe with a round, oval or cylindrical shape measuring 5-10 micrometres.The following is the classification of the Saccharomyces cerevisiae.
Saccharomyces cerevisiae tends to be sensitive to high-temperature pressure, and all yeast isolates cannot survive at temperatures of 40℃ and above.Fermented foods and beverages represent a prospective reservoir of microorganisms, encompassing probiotic organisms, fermentative microbes, or distinct microorganisms characterized by diverse physiological and genetic attributes.A study conducted by Lenka [9] using the traditional production of Brem Bali as a yeast isolate creates special environmental conditions that can support yeast growth to deal with severe stress conditions.Cultivation of Saccharomyces cerevisiae and Wickerhamomyces anomaly can positively influence the chemical composition and sensory features of alcoholic beverages.

Fermentation technology to produce Bali Brem
Brem is formed from a fermentation process where starch molecules in the material are broken down by fungi that produce amylolytic enzymes into dextrins and simple sugars such as disaccharides by scarification or enzymatic hydrolysis.Yeast will then convert some of these simple sugars into alcohol and CO2.The enzyme capable of converting glucose into alcohol and CO2 during fermentation is the zymase enzyme produced by the yeast Saccharomyces cerevisiae.
In the fermentation process, apart from alcohol, pyruvic acid and lactic acid are also formed.Pyruvic acid is an intermediate product formed in the hydrolysis of sugar to ethanol and can be converted into ethanol or lactic acid.In further fermentation, alcohol by Pediococcus sp. and Acetobacter sp.bacteria is oxidized to organic acids, including acetic acid, succinic acid, and malic acid.The conversion of pyruvic acid into lactic acid is catalyzed by the bacterium Pediococcus pentosaceus; through the alcohol oxidation process, some organic acids will react with alcohol to form esters which give the taste of tape [4].In making liquid brem, the black sticky tapai used in making liquid brem is placed in a specially designed container at the bottom so that the resulting water tapai can be collected.Tapai water is produced on the 2 nd to 4 th day of fermentation.The remaining tapai waste is used as a raw material for making snacks.During this period, the solids in the tapai water will settle so the brem becomes clear.The clear brem liquid is then carefully poured into bottles to be marketed.The water tape that has been collected is then left for seven months.
Generally, liquid brem is produced from fermented black glutinous rice, and with the addition of white glutinous rice, the alcohol content in liquid brem products can change during storage.The increase in alcohol content occurs due to the fermentation process, which continues during storage, while the decrease can be caused by esterification, oxidation and evaporation processes.Alcohol oxidation is due to the aerobic atmosphere that occurs during storage time.The aerobic atmosphere is usually followed by the activity of acetic bacteria so that acetic acid is formed, which makes the brem taste sour.In the storage process, if the product is not closed, alcohol evaporation will occur.The sour smell is caused by the formation of ethyl acetate ester from the reaction of alcohol with acetic acid formed by aerobic conditions and acetic bacteria.
Brem Bali, in its manufacture, uses natural ingredients; since 1976, the manufacturing process no longer uses preservatives or dyes (additives).This is done to maintain a truly distinctive taste.The product manufacturing process uses conventional biotechnology principles by utilizing microorganisms, namely the bacterium Saccharomyces cerevisiae.Purwanto and Agus [10] reported that the fermentation process in making liquid brem is the most important stage; there are four stages of decomposition in the process, which include (1) breaking down starch into dextrins and simple sugars; this process is an enzymatic hydrolysis process; (2) The sugar formed will be broken down into alcohol; (3) Furthermore, the alcohol formed is converted into organic acids by Pediococcus sp. and Acetobacter sp.bacteria through the oxidation process of alcohol; (4) Formation of a distinctive taste by some organic acids that react with alcohol, namely esters.The enzyme capable of converting glucose into alcohol and carbon dioxide during fermentation is the zymase enzyme produced by the yeast Saccharomyces cerevisiae.In the fermentation process, apart from alcohol, pyruvic acid and lactic acid are also formed.Pyruvic acid is an intermediate product formed in the hydrolysis of sugar to ethanol and can be converted into ethanol or lactic acid.The conversion of pyruvic acid to lactic acid is catalyzed by the bacterium Pediococcus pentosaceus.

The Important Role of Brem Bali for Health
Brem Bali, as one of the traditional fermented drinks, contains alcohol.The Ministry of Health of the Republic of Indonesia website reported that excessive alcohol consumption can cause a person to lose consciousness, have seizures, and even die.Apart from that, alcohol consumption can also cause diseases such as stomach ulcers, liver damage, and complications of severe psychiatric disorders.Most people assume that alcoholic beverages are negative, but various benefits can be generated if consumed within certain limits and levels.There are many alcoholic drinks, both locally and internationally, made in Bali, such as beer, wine, vodka, and so on, which are fermented products.In addition to lifestyle (lifestyle), there are also benefits for health.Higgins and Llanos [11] reported that fermented alcoholic beverages such as wine have the benefit of lowering cholesterol, reducing the risk of kidney stone disease, and helping to improve vision and improve skin elasticity.
Black glutinous rice (Oryza sativa glutinosa L.) is a glutinous rice used to manufacture traditional Balinese Brem drinks.Black glutinous rice has a color that tends to be deep purple and contains high phenolic compounds, especially anthocyanins.Glutinous rice grains are composed of aleurone, endosperm and embryo.In the aleurone and embryo, there are nutritional components in the form of vitamins (thiamin, riboflavin, and niacin), fat (linoleic, oleic, and palmitate), protein (oryzalin) and minerals (calcium, magnesium, iron, and phosphorus).In contrast, the endosperm is almost entirely starch [12].Black glutinous rice contains very high fibre, so it can potentially prevent the risk of heart disease and diabetes and helps improve digestion.Glutinous rice also contains antioxidants that benefit skin health and protect the body from harm.Antioxidants contained in glutinous rice can accelerate collagen production, which plays a role in skin elasticity.In addition, glutinous rice contains vitamins C, B6, B2, B1, and E, supporting skin health, boosting the immune system, and helping the body's metabolism run properly and healthily.

Problems and Challenges of Liquid Brem Fermentation
Rice wine is produced using traditional techniques, resulting in a product of inconsistent quality.However, it has been produced for centuries from generation to generation, and not much attention has been paid to improving its production methods [4].Tape yeast is used as an inoculum in production methods where liquefaction and alcoholic fermentation occur simultaneously without any attempt to control fermentation [2].The increase in alcohol content occurs due to the fermentation process, which continues during the storage process, while it will decrease due to esterification, oxidation and evaporation processes [4].Anisa and Bintoro [13], the higher the yeast concentration, the greater the alcohol content produced, the total dissolved solids, and the lower or decreased pH value.
The fermentation process in making Brem Bali drink made from glutinous rice produces alcohol with varying levels.Based on the Regulation of The Head of the Republic of Indonesia Drug and Food Control Agency No. 14 of 2016 concerning Safety and Quality Standards for Alcoholic Beverages in Chapter III Article 5 states that the maximum Methanol content in Alcoholic Beverages is no more than 0.01% v/v (calculated by product volume).Methanol is methyl alcohol with the chemical formula CH3OH which is commonly used as an extracting solvent and is toxic to humans.The group of alcoholic beverages in BPOM Regulations is divided into three based on alcohol content, namely group A (alcohol content up to 5%; group B (alcohol content more than 5-20%)); and group C (alcohol content more than 20-55 %).Brem circulating in the market has an alcohol content ranging from 3-10%, with the alcohol content contained in liquid brem products, which is certainly enough to be one of the obstacles in marketing for the body.Bishehsari et al. [14] reported that alcoholic beverages can affect the gut microbiota and intestinal inflammation.Consumption of alcoholic beverages is associated with disruption of gut microbiome homeostasis in rodent models and humans [15].The benefits of liquid brem as a traditional fermented drink are still not optimal because the alcohol content, they have can hurt the body.

Brem Bali Innovation and Industrialization
Bali brem or rice wine products can be modified into healthier alcoholic drinks.One of the developments is healthy brem with probiotics and durian peel flour.Probiotics, as officially defined by the Food and Agriculture Organization and the World Health Organization, refer to live microorganisms that, when administered in sufficient quantities, bestow health benefits upon consumers.Earlier report have observed that growing public recognition of the significance of maintaining a nutritious diet has spurred the growth of the health-focused food and beverage sector [16].Probiotic products have garnered increasing attention over time due to their potential to contribute to improvements in immunological, digestive, and respiratory health, along with their capacity to alleviate infectious diseases [17].
Additionally, Vasudha and Mishra asserted that probiotic milk stands as the sole available probiotic product, with its primary drawbacks being allergy and lactose intolerance.The preservation of probiotics poses a substantial challenge when applied to non-dairy products [18].In another study, Calumba et al. [19] proposed the potential of beer, the globally most consumed alcoholic beverage and the third most popular drink following water and tea, as a viable probiotic drink option.Calumba conducted research on the immobilization of Lactobacillus brevis bacteria in the alcoholic beverage Beer Ale.
Several investigations into the adaptation of probiotic alcoholic beverages can serve as valuable references for the development of fermented drinks like brem Bali or rice wine.Microorganisms, including probiotic bacteria, generally exhibit vulnerability to adverse conditions [20].Research has shown that alcohol can induce cell death by altering membrane fluidity and inhibiting microbial cell growth.However, gram-positive bacteria, particularly lactic acid bacteria, are recognized for their ability to thrive and multiply in high alcohol concentrations [21].The Lactobacillus genus, in particular, tends to exhibit considerable ethanol tolerance, capable of thriving in ethanol levels of up to 16% [22].Previous report demonstrated that the Lactobacillus brevis species utilized could flourish in an environment containing up to 3% butanol [23].
Worldwide beer production is estimated at 1.7 billion litres annually [24].This can be an opportunity for brem production to reach the world market by developing it into a healthier choice of alcoholic beverages with probiotics.Including probiotics in drinks containing alcohol has the potential to provide health benefits that cannot be provided by ordinary alcoholic drinks on the market.The study conducted by Alcine Chan et al. [25] aims to overcome the antimicrobial effect of hops iso-aacids by first co-fermenting the starter co-culture of Lactobacillus paracei L26 with Saccharomyces cerevisiae S-04 in uncut wort for ten days, in the presence of hops iso-a-acids in beer can be detrimental to the viability of probiotics.But in making brem drink you don't need hops.
Immobilization involves the confinement of substances within a matrix and has been demonstrated to enhance the viability and proliferation of probiotics in various products [26].Various immobilization supports have been explored, with a particular preference for biopolymers and natural supports that meet food-grade standards [26].One study reported the use of significant quantities of corn starch for the immobilization of probiotics [27].Calumba et al. [19] conducted research with the objective of producing probiotic ale beer, and in their study, they utilized durian powder as a means to support the immobilization of probiotic bacteria in the beer.Durian, a widely popular tropical fruit in Southeast Asia [28], is typically consumed with only a third of the fruit, while its fibrous skin constitutes more than half of its total weight [29,30].Teh et al. [31] assessed the potential of durian, mangosteen, and jackfruit peels as immobilizers for probiotics in soy milk.Additionally, the disposal of durian skin poses social and environmental challenges, making its use as an immobilizer a source of economic value.
Traditional beverages such as brem, arak, and Balinese palm wine are no longer restricted in terms of production and development Business licenses that can only be opened provide opportunities for industrialization and development of alcoholic beverages, one of which is brem while still paying attention to local wisdom.Brem artisans in Bali, such as MSMEs and even industrial establishments, can be established with a business permit that has been stipulated; production and marketing can be carried out legally while still following applicable regulations, such as capital, raw materials, standardization, to product marketing.The assistance for this business is carried out by the Cooperative Service to the Center for Drug and Food Control because there is a distribution of food consumption that needs to be met, such as requirements for alcohol content, process, and hygienic standards for brem products.

Study of Functional Properties of Brem Bali for Health
The International Food Information Council (IFIC) offers an official definition of functional food as food items with the potential to offer health advantages beyond fundamental nutrition.As per the regulatory authority, the Food and Drug Control Agency, functional food refers to items that have naturally undergone a specific process and contain one or more compounds supported by scientific research to possess physiological functions that promote health.Developing brem drinks with probiotics will have a good physiological impact on the body.The drink, made through the glutinous rice fermentation process, is believed to become a lifestyle for its connoisseurs and can also positively impact health.Higgins and Llanos [11] reported that fermented alcoholic beverages such as wine have the benefit of lowering cholesterol, reducing the risk of kidney stone disease, and helping to improve vision and skin elasticity.
Collagen that enters the body is broken down through a controlled process, namely hydrolysis, in which water molecules break the chemical bonds in collagen protein.Hydrolyzed collagen is broken down into small protein chains or collagen peptides of several amino acids.The body will absorb peptides and amino acids through the small intestine and then travel to support the body's natural collagen production through fibroblasts.Brem drink with the development of probiotics in it can provide health effects.Probiotics are living organisms that can provide beneficial effects on health when consumed in sufficient quantities [32,33] by improving the balance of intestinal microflora when they enter the digestive tract [34,35,36].
Probiotics confer health benefits to the body through three distinct functional mechanisms.Firstly, their protective function lies in their capacity to impede pathogens within the digestive tract.Probiotic colonization in the gastrointestinal system triggers competition for vital nutrients and adhesion sites with other bacteria, particularly pathogens.As probiotics flourish, they generate various antibacterial components, such as organic acids, hydrogen peroxide, and bacteriocins, which effectively suppress pathogenic growth [37,38].Secondly, probiotics enhance the body's immune system by stimulating the production of IgA and activating macrophages, modulating cytokine profiles, and inducing a state of reduced responsiveness to food-derived antigens.Thirdly, probiotic metabolites play a crucial role, including the degradation of lactose in fermented milk products, making them suitable for individuals with lactose intolerance [37].These are the observed effects of probiotics on health and the underlying mechanisms.
Probiotics, by inducing the production of B-defensin and IgA from the host, bring about alterations in the composition of the intestinal microbiota, effectively inhibiting the proliferation of pathogens.Moreover, probiotics contribute to the reinforcement of the intestinal barrier by preserving tight junctions and stimulating mucin production.Additionally, probiotics serve as immunomodulators by influencing signaling pathways like NF-kB and MAP kinases, thereby impacting the proliferation and differentiation of immune cells and epithelial cells [39,40].

Conclusion
Brem Bali, a traditional Balinese drink, one of the facilities in religious ceremonies, is now widely known as a welcome drink and souvenir.Brem products must be innovated as a traditional fermented beverage for better development and market acceptance.One of the innovations that can be made in the development of brem beverage products into alcoholic beverages with the addition of probiotics.The selection of durian peel flour is an alternative to immobilizing probiotic bacteria in brem products because it can help the country's economy and handle durian peel waste.Brem Bali, as a fermented drink, besides being able to provide probiotic benefits, also acts as an immunomodulator.It maintains digestive health by preventing irritable bowel syndrome, constipation, non-pathogenic diarrhoea, flatulence, lactose intolerance, cariogenic, carcinogenic, mutagenic tumours, intestinal inflammation, and growth of intestinal bacteria.Excess lower blood cholesterol and reduce hypertension.The implementation of Presidential Regulation No. 10 of 2021 related to the Investment Business Sector reinforces the provisions outlined in Bali Governor Regulation No. 1 of 2020 concerning the Supervision of Fermented and Distilled Beverages in Bali.This regulation has granted permits for industrial businesses and the expansion of Balinese fermented beverage businesses to be developed by the community.Business licenses that can only be opened provide opportunities for industrialization and development of alcoholic beverages, one of which is brem while still paying attention to local wisdom.Brem artisans in Bali, such as MSMEs and even industrial establishments, can be established with a business permit that has been stipulated; production and marketing can be carried out legally while still following applicable regulations, such as capital, raw materials, standardization, to product marketing.The Cooperative Service assists the Center for Drug and Food Control in this effort because food consumption distribution needs to be met, such as requirements for alcohol content, process and hygienic standards for Brem products.
. The issuance of Presidential Regulation Number 10 of 2021 concerning the Investment Business Sector reinforces Bali Governor Regulation Number 1 of 2020 regarding the Management of Balinese Fermented and Distilled Beverages.These established regulations grant permits for industrial enterprises and facilitate the expansion of Balinese fermented and distilled beverage industries, originally intended for religious rituals, but now open to extensive development by the Balinese community.